Problem with ligation..! - (Apr/10/2008 )

Hi all,

I am desperately seeking help regarding these purification kits. I am trying to clone now. I restriction digested my plasmid and PCR insert seperately in a 50 micro litre reaction volume.Then I purified the vector using the Quiaprep kit and the insert using Quiaquick PCR purification kit.

My yield after purification is rather shocking to me. My vector, eluted in 50 micro litre shows just 8 ng/ micro litre (in nano drop )The insert shows 20 ng/microlitre. It seems like the DNA just disappears. I am very worried. Could someone suggest what could have gone wrong. Also how much of insert and vector is needed for a 10 micro litre ligation reaction (5:1 ratio)

Please send me ur views!!!

-lp148-

How much material did you start with?

-zek-

i started with vector 193 ng / micro lit and insert after pcr purification about 100 ng/micro lit

-lp148-

I mean how many ug of vector and insert did you use for restriction?I have not used the PCR purification kit from Qiagen but I use the Gelextraction Kit (500). Sometimes the recovery of DNA is very low. So, it`s good to start with 2-3 ug DNA for restriction.

For regular ligation 50ng of vector and the corresponding amount of insert should be enough (although I don`t know about the details of your experiment)

-zek-

We have similar problems with gel extraction kits. The QIAGEN rep suggested I heat the elution buffer to 65C, and incubate it on the filter for 1 minute before centrifuging. This did make a significant difference, but the yields are still ~60%.

-swanny-

Hallo I had bad experiences with this qiagen kits as well, 900ng per ul PCR prroduct after purificatio resulted in 60ng per ul. There is one posibillity, Digestion of pcr product is insuficient if recognition sites are on the ends of the product. If it is like this ligate PCR product before digest, it create concatenate, a better substrate for restriction digestion and than ligate it to vector. Or prepare ligation reation in Volume 20-30ul(whole mixture can by used for transformation E coli), most of reaction counpounds do not inhibit ligase and if 1-2ul of PCR product and vector will by used, dilution will by around 15X so it should by ok. But by sure restrictase was right inactivated by tempriture!!!!OR:Do not use kit for purification but do it old way, ethanol precipitation. Ligation ratio should by 1:3 vector insert, since ligation is bimoleculaar reaction ,but 1:5 increase chance for succesreply me if it solve your problemsgood luck